Biological Function of IL-10
IL-10 is an α-helical cytokine that is expressed as a non-covalently linked homodimer of ˜37 kDa. It plays a key role in the induction and maintenance of tolerance. Its predominantly anti-inflammatory properties have been known for a long time. IL-10 suppresses the secretion of pro-inflammatory cytokines like TNF α, IL-1, IL-6, IL-12 as well as Th1 cytokines such as IL-2 and INFγ and controls differentiation and proliferation of macrophages, B-cells and T-cells (Glocker, E. O. et al., Ann. N.Y. Acad. Sci. 1246, 102-107 (2011); Moore, K. W. et al., Annu. Rev. Immunol. 19, 683-765 (2001); de Waal Malefyt, R. et al., J. Exp. Med. 174, 915-924 (1991); Williams, L. M. et al., Immunology 113, 281-292 (2004). Moreover, it is a potent inhibitor of antigen presentation, inhibiting MHC II expression as well as upregulation of co-stimulatory molecules CD80 and CD86 (Mosser, D. M. & Yhang, X., Immunological Reviews 226, 205-218 (2008)).
Nevertheless, also immunostimulatory properties have been reported. IL-10 can costimulate B-cell activation, prolong B-cell survival, and contribute to class switching in B-cells. Moreover, it can costimulate natural killer (NK) cell proliferation and cytokine production and act as a growth factor to stimulate the proliferation of certain subsets of CD8+ T cells (Mosser, D. M. & Yhang, X., Immunological Reviews 226, 205-218 (2008); Cai, G. et al., Eur. J. Immunol. 29, 2658-2665 (1999); Santin, A. D. et al., J. Virol. 74, 4729-4737 (2000); Rowbottom, A. W. et al., Immunology 98, 80-89 (1999); Groux, H. et al., J. Immunol. 160, 3188-3193 (1998)). Importantly, high doses of IL-10 (20 and 25 μg/kg, respectively) in humans can lead to an increased production of INFγ (Lauw, F. N. et al., J. Immunol. 165, 2783-2789 (2000); Tilg, H. et al., Gut 50, 191-195 (2002)).
IL-10 signals through a two-receptor complex consisting of two copies each of IL-10 receptor 1 (IL-10R1) and IL-10R2. IL-10R1 binds IL-10 with a relatively high affinity (˜35-200 pM) (Moore, K. W. et al., Annu. Rev. Immunol. 19, 683-765 (2001)), and the recruitment of IL-10R2 to the receptor complex makes only a marginal contribution to ligand binding. However, the engagement of this second receptor to the complex enables signal transduction following ligand binding. Thus, the functional receptor consists of a dimer of heterodimers of IL-10R1 and IL-10R2. Most hematopoietic cells constitutively express low levels of IL-10R1, and receptor expression can often be dramatically upregulated by various stimuli. Non-hematopoietic cells, such as fibroblasts and epithelial cells, can also respond to stimuli by upregulating IL-10R1. In contrast, the IL-10R2 is expressed on most cells. The binding of IL-10 to the receptor complex activates the Janus tyrosine kinases, JAK1 and Tyk2, associated with IL-10R1 and IL-10R2, respectively, to phosphorylate the cytoplasmic tails of the receptors. This results in the recruitment of STAT3 to the IL-10R1. The homodimerization of STAT3 results in its release from the receptor and translocation of the phosphorylated STAT homodimer into the nucleus, where it binds to STAT3-binding elements in the promoters of various genes. One of these genes is IL-10 itself, which is positively regulated by STAT3. STAT3 also activates the suppressor of cytokine signaling 3 (SOCS3), which controls the quality and quantity of STAT activation. SOCS3 is induced by IL-10 and exerts negative regulatory effects on various cytokine genes (Mosser, D. M. & Yhang, X., Immunological Reviews 226, 205.218 (2008)).
Genetic linkage analyses and candidate gene sequencing revealed a direct link between mutations in IL-10R1 and IL-10R2 and early-onset enterocolitis, a form of inflammatory bowel disease (IBD) (Glocker, E. O. et al., N. Engl. J. Med. 361(21), 2033-2045 (2009)). Recent data suggest that early onset IBD can even be monogenic. Mutations in the IL-10 cytokine or its receptors lead to a loss of IL-10 function and cause severe enterocolitis in infants and small children (Glocker, E. O. et al., Ann. N.Y. Acad. Sci. 1246, 102-107 (2011)). Moreover, patients with severe forms of Crohn's disease have a defective IL-10 production in whole blood cell cultures and monocyte-derived dentritic cells (Correa, I. et al., J. Leukoc. Biol. 85(5), 896-903 (2009)). IBD affects about 1.4 million people in the United States and 2.2 million in Europe (Carter, M. J. et al., Gut 53 (Suppl. 5), V1-V16 (2004); Engel, M. A. & Neurath, M. F., J. Gastroenterol. 45, 571-583 (2010)).
Therapeutic Approaches Using IL-10
The therapeutic benefit of recombinant IL-10 in inflammatory disorders and autoimmune disease has been assessed in phase I & II clinical trials investigating safety, tolerance, pharmacokinetics, pharmacodynamics, immunological and hematological effects of single or multiple doses administered intravenously or subcutaneously in various settings on healthy volunteers as well as specific patient populations (Moore, K. W. et al., Annu. Rev. Immunol. 19, 683-765 (2001); Chernoff, A. E. et al., J. Immunol. 154, 5492-5499 (1995); Huhn, R. D. et al., Blood 87, 699-705 (1996); Huhn, R. D. et al., Clin. Pharmacol. Ther. 62, 171-180 (1997)). IL-10 was well tolerated without serious side effects at doses up to 25 μg/kg and only mild to moderate flu-like symptoms were observed in a fraction of recipients at doses up to 100 μg/kg (Moore, K. W. et al., Annu. Rev. Immunol. 19, 683-765 (2001); Chernoff, A. E. et al., J. Immunol. 154, 5492-5499 (1995)). Tendencies towards clinical improvement were most often seen in psoriasis (a compilation of clinical studies can be found in Mosser, D. M. & Yhang, X., Immunological Reviews 226, 205-218 (2008)), Crohn's disease (Van Deventer S. J. et al, Gastroenterology 113, 383-389 (1997); Fedorak, R. N. et al., Gastroenterology 119, 1473-1482 (2000); Schreiber, S. et al., Gastroenterolotgy 119, 1461-1472 (2000); Colombel J. F. et al., Gut 49, 42-46 (2001)) and rheumatoid arthritis (Keystone, E. et al., Rheum. Dis. Clin. N. Am. 24, 629-639 (1998); Mosser, D. M. & Yhang, X., Immunological Reviews 226, 205-218 (2008)).
Overall, the clinical results were unsatisfying and clinical development of recombinant human IL-10 which is identical to endogenous human IL-10 with the exception of a methionine residue at the amino terminus (ilodecakin, TENOVIL, Schering-Plough Research Institue, Kenilworth, N.J.) was discontinued due to a lack of efficacy. A recent systematic review of the efficacy and tolerability of recombinant human IL-10 for induction of remission in Crohn's disease found no statistically significant differences between IL-10 and placebo for complete or clinical remission and stated that patients treated with IL-10 were significantly more likely to withdraw from the studies due to adverse events relative to placebo (Buruiana, F. E. et al., Cochrane Database Syst. Rev. 11, CD005109 (2010)) For Crohn's disease, several reasons for these unsatisfying results have been discussed (Herfarth, H. & Schölmerich, J., Gut 50, 146-147 (2002)): 1) local cytokine concentrations in the gut that were too low to mediate a sustained anti-inflammatory effect, 2) dose escalation of systemically administered IL-10 was limited due to side effects, and 3) the immunostimulatory properties of IL-10 on B cells and on INFγ production by CD4+, CD8+, and/or natural killer cells counterbalance its immunosuppressive properties (Asadullah, K. et al., Pharmacol. Rev. 55, 241-269 (2003); Tilg, H. et al., Gut 50, 191-195 (2002); Lauw, F. N. et al., J. Immunol. 165, 2783-2789 (2000)).
IL-10 exhibits a very short plasma half-life due to its small size of ˜37 kDa which leads to rapid kidney clearance. In fact, its half life in the systemic compartment is 2.5 h which limits the mucosal bioavailability (Braat, H. et al., Expert Opin. Biol. Ther. 3(5), 725-731 (2003). In order to improve circulation time, exposure, efficacy and to reduce renal uptake, several publications report the PEGylation of this cytokine (Mattos, A. et al., J. Control Release 162, 84-91 (2012); Mumm, J. B. et al., Cancer Cell 20(6), 781-796 (2011); Alvarez, H. M. et al., Drug Metab. Dispos. 40(2), 360-373 (2012)). Nevertheless, the longer systemic half-life of PEGylated non-targeted IL-10 can exacerbate known adverse events of this molecule.
It has become clear that systemic treatment using recombinant human IL-10 is not sufficiently effective and that the focus has to be on local delivery of the cytokine. There are several ways to achieve this goal: 1) IL-10 gene therapy of immune cells, 2) genetically modified, non-pathogenic, IL-10 expression bacteria and 3) antibody-IL-10 fusion proteins in order to target the cytokine to and to accumulate the cytokine in inflamed tissues.
IL-10 gene therapy of immune cells has demonstrated effectiveness in experimental colitis but clinical trials are hampered by concerns over the safety of this approach for non-lethal diseases (Braat, H. et al., Expert Opin. Biol. Ther. 3(5), 725-731 (2003)). Transgenic bacteria (Lactococcus lactis) expressing IL-10 represent an alternative route of delivery and the outcome of a phase I trial in Crohn's disease was published claiming to avoid systemic side effects due to local delivery into the mucosal compartment and to be biologically contained (Braat, H. et al., Gastroenterol. Hepatol. 4, 754-759 (2006); Steidler, L. et al., Science 289, 1352-1355 (2000)). A phase IIa randomized placebo-controlled double-blind multi-center dose escalation study to evaluate the safety, tolerability, pharmacodynamics and efficacy of genetically modified Lactococcus lactis secreting human IL-10 (AG011, ActoGeniX) in patients with moderately active ulcerative colitis was well-tolerated and safe. However, there was no significant improvement of mucosal inflammation, as measured by the modified Baron score, or clinical symptoms in patients receiving AG011 compared with placebo (Vermeire, S. et al., abstract 46 presented at the Digestive Disease Week Annual Meeting in New Orleans 2 May 2010).
Antibody-cytokine fusion proteins, also called immunocytokines, offer several advantages in terms of drug delivery and the format of the drug itself. Local delivery of cytokines, e.g. IL-10, is achieved by fusion to antibodies or fragments thereof specific for suitable disease markers. Thus, systemic side effects can be reduced and local accumulation and retention of the compound at the site of inflammation can be achieved. Moreover, depending on the fusion format and antibody or antibody fragment used, properties like plasma half-life, stability and developability can be improved. Although an already established approach in oncology, it was only recently adapted in order to treat inflammatory disorders and autoimmunity. Several cytokines (IL-10 amongst others) and a photosensitizer were targeted to psoriatic lesions by fusion to a scFv antibody fragment specific for the extra domain B of fibronectin (Trachsel, E. et al., J. Invest. Dermatol. 127(4), 881-886 (2007). Moreover, antibody fragments specific for the extra domain A of fibronectin (F8, DEKAVIL, Philogen SpA)-IL-10 fusion proteins were used preclinically to inhibit the progression of established collagen-induced arthritis (Trachsel, E. et al., Arthritis Res. Ther. 9(1), R 9 (2007); Schwager, K. et al., Arthritis Res. Ther. 11(5), R142 (2009)) and entered clinical trials. Recently, the same F8-IL-10 fusion protein was used for targeting endometriotic lesions in a syngeneic mouse model and reduced the average lesion sizes compared to the saline control group (Schwager, K. et al., Hum. Reprod. 26(9), 2344-2352 (2011)).
The IgG-IL-10 fusion proteins of this invention have several advantages over the known antibody fragment-based (e.g. scFv, diabody, Fab) IL-10 fusion proteins, including improved produceability, stability, serum half-life and, surprisingly, significantly increased biological activity upon binding to target antigen.